A variety of methods are currently available for making arrays of biological macromolecules, such as arrays of nucleic acid molecules or proteins. One method for making ordered arrays of DNA on a porous membrane is a “dot blot” approach. In this method, a vacuum manifold transfers a plurality, e.g., 96, aqueous samples of DNA from 3 millimeter diameter wells to a porous membrane. A common variant of this procedure is a “slot-blot” method in which the wells have highly-elongated oval shapes. The DNA is immobilized on the porous membrane by baking the membrane or exposing it to UV radiation. This is a manual procedure practical for making one array at a time and usually limited to 96 samples per array. “Dot-blot” procedures are therefore inadequate for applications in which many thousand samples must be determined.
An alternate method of creating ordered arrays of nucleic acid sequences is described by Pirrung, et al. (U.S. Pat. No. 5,143,854, 1992), and also by Fodor, et al. (Science 251:767-773, 1991). The method involves synthesizing different nucleic acid sequences at different discrete regions of a support. This method employs elaborate synthetic schemes, and is generally limited to relatively short nucleic acid samples, e.g., less than 20 bases. A related method has been described by Southern, et al. (Genomics 13:1008-1017, 1992).
Montgomery (U.S. Pat. No. 6,093,302, 2000) teaches a method for making arrays of polymers by employing electrochemically generated reagents that are confined by scavenging/buffering agents. Synthesis of the polymers occurs on a microarray of chemically-modified electrodes by passing current through subsets of the electrodes (the active electrodes) to produce the electrochemically-generated reagents (EGR) locally, near the electrodes. The scavenging agent is present in solution prior to passing current through the electrodes. The concentration of this scavenging agent must be carefully balanced so that it does not prevent synthesis locally at the desired subset of electrodes but can eliminate any unwanted reactions at the remaining electrodes where subsequent rounds of synthesis will occur (the passive electrodes). As the density of electrodes increases, the distance between active and passive electrodes decreases, and the proper balance of EGR and scavenger becomes problematic. This balance is best understood as a high concentration of EGR near the active electrodes and a high concentration of scavenger at some distance from these electrodes. In between these two areas of high concentration will be a gradient of the two, opposing agents. This gradient is established solely by the active electrode since the scavenger is present in solution everywhere at a fixed concentration prior to passing current. It is expected that partial synthesis could occur in this gradient zone. As electrode densities increase, the passive electrodes could fall within this zone, thereby leading to unwanted reactions and poor overall synthesis.
Southern & Egeland (U.S. patent application Ser. No. 10/488,058) describe an electrochemical method for producing microarrays that utilizes an array of electrodes but differs from Montgomery (U.S. Pat. No. 6,093,302, 2000) in several important ways. First, the electrodes are thin lines (0.75 cm×40 um) and do not behave as approximate point sources like true microelectrodes do. This electrode configuration only allows the synthesis of polymers (e.g. oligonucleotides) with highly constrained sequences of monomers. That is, one cannot synthesize a microarray (˜40 um×40 um feature size) with any sequence of monomers at any position in the array. Second, the electrochemical reactions that produce the microarray are physically limited to a thin layer of solvent (˜40 um) over the surface of the array substrate. This limitation imposes complicated mechanical requirements on fluid delivery of synthesis reagents to the microarray. Third, a scavenger is used to confine the reaction to the active electrodes in a manner analogous to Montgomery (U.S. Pat. No. 6,093,302, 2000). However, Southern & Egeland (U.S. patent application Ser. No. 10/488,058) produce this scavenger by electrochemical means at electrodes on the array that surround the active electrodes. In effect, these “surrounding electrodes” produce a wall of the scavenging agent that neutralizes the EGR thereby preventing the EGR from diffusing to the passive electrodes. The requirement for surrounding electrodes limits the geometry of the electrode array.
Montgomery (U.S. Pat. No. 6,093,302, 2000) also describes the use of a second set of electrodes, called the “getter” structure, which functions actively to scavenge the EGR. This function is performed by applying sufficient potential to the “getter” structure to cause electrochemical scavenging. The “getter” structure is distinct from those electrodes where synthesis occurs. Similar to Southern & Egeland (U.S. patent application Ser. No. 10/488,058), the “getter” structure is a “surrounding” electrode and prevents the EGR made at the active electrodes from reaching the passive electrodes. Montgomery describes the preferred form of the “getter” structure as “ring” electrodes that surround the electrodes where synthesis occurs. This separate “getter” structure adds complexity to the array design, and limits the geometry of the electrode array thereby impacting its achievable density.
There is a need in the art for a method of synthesizing flexible configurations of polymers by electrochemical means on high density arrays of chemically-modified electrodes. The current invention addresses this need by using electrochemical means to generate the scavenging agent at the passive electrode. In this way, a “virtual cap” is created directly over the passive electrodes thereby preventing the EGR from causing unwanted reactions.